As used throughout this specification, the following definitions apply for purposes of the present invention:
The term "expression" may be characterized in the following manner. A cell is capable of synthesizing many proteins. At any given time, many proteins which the cell is capable of synthesizing are not being synthesized. When a particular polypeptide, coded for by a given gene, is being synthesized by the cell, that gene is said to be expressed. In order to be expressed, the DNA sequence coding for that particular polypeptide must be properly located with respect to the control region of the gene. The function of the control region is to permit the expression of the gene under its control.
The term "vector" refers to an extra-chromosomal molecule of duplex DNA comprising an intact replicon that can be replicated in a cell. Generally, vectors are derived from viruses or plasmids of bacteria and yeasts. A baculovirus vector comprises a baculovirus replicon.
The term "gene" refers to those DNA sequences which transmit the information for and direct the synthesis of a single protein chain.
The term "infection" refers to the invasion by agents (e.g., viruses, bacteria, etc.) of cells where conditions are favorable for their replication and growth.
The term "transfection" refers to a technique for infecting cells with purified nucleic acids of viruses.
The term "heterologous gene" in reference to the baculovirus vectors hereof, refers to DNA that encodes polypeptides ordinarily not produced by the virus from which the vector is derived, but which is introduced into the cell as recombinant DNA or within viruses carrying recombinant DNA genomes. The terms "passenger gene" or "passenger DNA" as used herein are equivalent to the term "heterologous gene." "Exogenous," as used herein, has the same meaning as heterologous.
The term "transplacement plasmid" means a bacterial vector which is used as an intermediate in the construction of a virus vector. A transplacement plasmid facilitates the transfer of exogenous genetic information, such as the combination of a novel promoter and a heterologous structural gene under the regulatory control of that promoter, to a specific site within the viral genome by homologous recombination. That homologous recombination occurs via the DNA sequences flanking the chimeric gene.
The science of genetic engineering has advanced to a stage wherein certain biologically useful products can be produced in large quantities. For example, two commercially successful drugs, human growth hormone and tissue plasminogen activator (t-PA), are now being produced in large quantities and are being used to treat a variety of pathological conditions. However, scientists are constantly trying to discover new and more efficient systems for producing the proteins and other products in various biological systems.
The technology of transferring genes from one species and expressing them in another is made possible because the DNA of all living organisms is chemically similar in that it is composed of long chains containing the same four nucleotides. Nucleotide sequences are arranged in codons (triplets) which code for specific amino acids with the coding relationship between the amino acid and nucleotide sequence being essentially the same for all species of organisms. The DNA is organized into genes which are comprised of control regions which mediate initiation of expression of the gene and coding regions. These control regions are commonly referred to as "promoters." An enzyme, called RNA polymerase, binds to the promoter region and is either activated or in some way is signalled so that it travels along the coding region and transcribes the encoded information from the DNA into messenger ribonucleic acid (mRNA). The mRNA contains recognition signals: signals for ribosome binding, signals for translational start and stop, and for polyadenylation. Cellular ribosomes then translate the nucleotide codon information of the mRNA into protein with an amino acid sequence specified by the nucleotide codon sequence.
The general use of restriction endonucleases and the ability to manipulate DNA sequences has been greatly improved by the availability of chemically synthesized double stranded oligonucleotides containing desired nucleotide sequences including useful restriction site sequences. Virtually, any naturally occurring, cloned, genetically altered or chemically synthesized segment of DNA can be coupled to any other segment by attaching an oligonucleotide containing the appropriate sequences or recognition sites to the ends of the DNA molecule. Subjecting this product to the hydrolytic action of the appropriate restriction endonuclease produces the requisite complementary ends for coupling the DNA molecules. While there are many possible variations in gene transfer schemes, it is important to note that the techniques are available for inserting DNA sequences in the proper location and orientation with respect to a promoter region to allow expression of those sequences.
Potentially, any DNA sequence can be inserted into a vector molecule to construct an artificial recombinant molecule or composite, sometimes called a chimera or hybrid DNA. For most purposes, the vector utilized is a duplex extra-chromosomal DNA molecule comprising an intact replicon such that the recombinant DNA molecule can be replicated when placed into bacteria or yeast by transformation. Vectors commonly in use are derived from viruses or plasmids associated with bacteria and yeast.
Because of the nature of the genetic code, the inserted gene or portions thereof will direct the production of the amino acid sequence for which it codes if the gene or gene portion is attached to a control region (promoter) which is capable of regulating expression in the cell in which the vector replicates. The general techniques for constructing expression vectors with cloned genes located in the proper relationship to promoter regions are described in the literature (e.g., See T. Maniatis, et al. (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
A number of vector systems utilizing the above-described general scheme and techniques have been developed for use in the commercial or experimental synthesis of proteins by genetically modified organisms. Many of these vector systems utilize prokaryotic bacterial hosts for vector replication and heterologous gene expression. Additionally, systems have been utilized which employ eukaryotic cells for vector replication and heterologous gene expression. Such systems are employed for hepatitis B virus surface antigen synthesis and for human tissue plasminogen activator synthesis.
Eukaryotic hosts are preferred for the production of some eukaryotic proteins which require modification after synthesis (i.e., glycosylation) to become biologically active. Prokaryotic cells are generally incapable of such modifications.
The use of virus vectors in eukaryotic hosts has been the subject of a considerable amount of recent investigation. Viral vector systems may suffer from significant disadvantages and limitations which diminish their utility. For example, some viral vectors are not able to achieve high enough levels of gene expression for economic protein production in costly eukaryotic cell culture systems. Some eukaryotic viral vectors are either pathogenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with accidental infection. Some virus vectors have severe limitations on the size of the heterologous gene that can be stably inserted into the virus particles.
As the genetic engineering technology becomes more sophisticated, there will be an increased interest in inserting more than one heterologous gene, i.e., genes coding for more than one protein, into a host cell to achieve coordinated expression and possibly to obtain coordinated activity of the various gene products.
An ideal viral vector should be capable of stably carrying a large segment of heterologous DNA, efficiently infecting cells and converting virtually all the protein biosynthesis of the infected cell to the high level expression of the foreign gene. A virus that appears to be well suited as a vector for the propagation and high level expression of many heterologous genes in a higher eukaryotic environment is the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). (See Miller, L. K. (1981) "Virus Vector for Genetic Engineering in Invertebrates," In Genetic Engineering in the Plant Sciences, N. Panopolous (ed.), Praeger Publ., N.Y., pp. 203-224; U.S. Pat. No. 4,745,051.)
The baculovirus group includes the subgroups of nuclear polyhedrosis viruses (NPV) and granulosis viruses (GV); baculoviruses infect only arthropod hosts. The virus particles of NPV and GV are occluded in proteinaceous crystals. In occluded forms of baculoviruses, the virions (enveloped nucleocapsids) are embedded in a crystalline protein matrix. This structure, referred to as an inclusion or occlusion body, is the form found extraorganismally in nature and is responsible for spreading the infection between organisms. The subgroup NPV produces many virions embedded in a single, large (up to 5 micrometers) polyhedral crystal, whereas the subgroup GV produces a single virion embedded in a small crystal. The crystalline protein matrix in either form is primarily composed of a single 25 to 33 kDa polypeptide which is known as polyhedrin or granulin in NPV or GV, respectively.
More general information on the subject of baculovirus structure and the process of infection is available in the following reviews: Carstens (1980) "Baculoviruses--Friend of Man, Foe of Insects? ," Trends and Biochemical Science, 52:107-10; Harrap and Payne (1979) "The Structural Properties and Identification of Insect Viruses" in Advances in Virus Research, Vol. 25, M. A. Lawer et al. (eds.), Academic Press, New York, pp. 273-355; and Miller, L. K. (1981) supra.
Baculovirus helper-independent viral vectors are particularly useful for the high-level production of biologically active eukaryotic proteins. Expression levels for some foreign genes have been reported to be 10% to 25% of the total protein of the recombinant infected cell. Appropriate post-translational modifications, including signal peptide cleavage, glycosylation, phosphorylation, oligomerization, complex formation, isolation and proteolysis, have been reported for a variety of different heterologous proteins produced using this expression system.
The baculovirus expression vectors described to date use very late promoters, such as the polyhedrin or polypeptide 10 (p10) promoters to drive foreign gene expression. (Reviewed by Luckow and Summers (1988) "Trends in the Development of Baculovirus Expression Vector," Bio/Technology :47-55 Miller, L. K. (1988) "Baculoviruses as Gene Expression Vectors," Ann. Review of Microbiology, 42:177-199.) These promoters are regulated during the course of virus infection and are activated very late in the infectious process usually beginning 18 to 24 hours post-infection. The polyhedrin and p10 genes are not essential for replication in cell culture, so the gene can be replaced with the heterologous gene of interest without interfering with the production of the budded form of the virus. The replacement of the polyhedrin gene, however, does interfere with the formation of the occluded form of the virus. The absence of the occluded virus in recombinant plaques provides a useful although somewhat tedious phenotypic selection for the recombinant viruses. It also places limitations on the ability to use the recombinant viruses for less expensive mass protein production in insect larvae.
The economic value and general utility of a baculovirus vector is strongly dependent on the nature of the promoter used to drive heterologous gene expression. The polyhedrin promoter is currently the promoter of choice for the production of high levels of protein. However, even higher levels of protein production are often necessary for economic feasibility. In addition to the need for high productivity, vectors are needed which can express more than one heterologous gene. The disadvantage to such vectors is that they are often genomically unstable if promoter sequences are duplicated within the vector. To minimize such instability, a different promoter must be employed for each heterologous gene to be expressed. These promoters must be different (nonhomologous) from naturally occurring viral promoters within the vector to more fully avoid genomic instability problems. Vectors should also have the ability to include promoters that express genes at an earlier stage of protein production to improve protein quality and to ensure that the protein has the necessary post-translational modifications to confer biological activity or immunological authenticity. Such post-translational modifications appear to decline during the very late phase of infection when the very late promoters of AcMNPV vectors, such as polyhedrin and p 10, are activated.
What are needed are modified or synthetic promoter regions which will cause significantly increased expression of the heterologous gene in the baculovirus system or which will allow the production of high quality protein. The new promoters should be flexible enough so that either a single heterologous gene or a series of heterologous genes can be inserted into the vector system so that various proteins can be produced at the same or at different times. In addition, the polyhedrin gene should be present if the virus is to be administered orally to the appropriate insect host.